Responsive Accumulation of Nanohybrids to Boost NIR‐Phototheranostics for Specific Tumor Imaging and Glutathione Depletion‐Enhanced Synergistic Therapy

Abstract Dynamic regulation of nanoparticles in a controllable manner has great potential in various areas. Compared to the individual nanoparticles, the assembled nanoparticles exhibit superior properties and functions, which can be applied to achieve desirable performances. Here, a pH‐responsive i‐motif DNA‐mediated strategy to tailor the programmable behaviors of erbium‐based rare‐earth nanoparticles (ErNPs) decorated copper doped metal‐organic framework (CPM) nanohybrids (ECPM) under physiological conditions is reported. Within the acidic tumor microenvironment, the i‐motif DNA strands are able to form quadruplex structures, resulting in the assembly of nanohybrids and selective tumor accumulation, which further amplify the ErNPs downconversion emission (1550 nm) signal for imaging. Meanwhile, the ECPM matrix acts as a near‐infrared (NIR) photon‐activated reactive oxygen species (ROS) amplifier through the singlet oxygen generation of the matrix in combination with its ability of intracellular glutathione depletion upon irradiation. In short, this work displays a classical example of engineering of nanoparticles, which will manifest the importance of developing nanohybrids with structural programmability in biomedical applications.


General Information
Materials. Tetrakis(4-carboxyphenyl) porphyrin, polyvinylpyrrolidone (MW 40000) and singlet oxygen sensor green (SOSG) were purchased from VWR international. Characterization. Transmission electron microscopy (TEM) images were acquired on the FEI Tecnai T12 electron microscope. Elemental mapping was acquired on the scanning transmission electron microscope (JEM-3200FS, JEOL, Japan). The hydrodynamic diameter distribution and zeta potential of various MOF NPs were measured on a scientific dynamic light scattering nanoparticle analyzer (SZ-100, Horiba). The XRD pattern was recorded on a D8 Advance diffractometer (Bruker, Germany). UV-Vis absorption spectrum was recorded on a Shimadzu UV-2501 spectrophotometer. Confocal laser scanning microscopy (CLSM) images was recorded on a Zeiss LSM 780 microscope. Flow cytometry analysis was carried out on a BD Beckman Coulter flow cytometer (Brea, CA). H&E tissue and cell staining was performed by BBC Biochemical (Mount Vernon, WA) and the images were collected using a BX41 bright field microscopy (Olympus). PET images were recorded on an Inveon smallanimal PET scanner system (Siemens Medical Solutions, USA). NIR-II images were acquired on home-made equipment. 1-3
After the reaction was done, the product was collected by centrifugation at 12 000 rpm for 10 min followed by washing with fresh DMF three times, which was then re-dispersed in 10 mL of DMF for further use.
Synthesis of core NaGdF 4 :10%Er nanoparticles. The small hexagonal phase (β-) NaGdF4:10%Er nanoparticles were synthesized following a previously reported method. 4 In a typical procedure, GdCl 3 ·6H 2 O (334.53 mg, 0.9 mmol), ErCl 3 ·6H 2 O (38.17 mg, 0.1 mmol), oleic acid (OA, 6.0 mL) and octadecene (ODE, 15.0 mL) were mixed together and heated to 160 °C with vigorous stirring under argon for 60 min to form a homogeneous transparent solution, and then solution was cooled down to room temperature. A methanol solution (10.0 mL) of ammonium fluoride (0.148 g, 4 mmol) and sodium hydroxide (0.1 g, 2.5 mmol) was added drop by drop and stirred for 1 h. The solution was then slowly heated and degassed at 110 °C for 20 min and then refilled with argon and degassed three times in total. After that the solution was heated to 280 °C within 10 min and reacted for 60 min under argon. After the solution was cooled back to room temperature, the products were precipitated from the solution with ethanol and washed with ethanol and water (1:1) three times. The nanoparticles were finally dispersed in 10 mL of cyclohexane for further use.

Study Approval
The procedures for the animal experiments were implemented under protocols of the National

GSH concentration in cells after various treatments. The intracellular consumption of GSH was
detected by GSH assay kit. Briefly, U87 cells were seeded into 6-well plates for 24 h (37 °C, 5% CO 2 ).
Then, the cells were treated as following: (1) Control, (2) NIR, (3) ECPMD1, (4) ECPMD2+NIR and (5) ECPMD1+NIR. After 24 h incubation, group (4) and (5)        As showed in Figure S6, the ErNPs displayed a typical absorption peak within 960-990 nm, which was stronger than that around 808 nm. Therefore, the ErNPs demonstrated stronger UCL and DLC emission intensities under 980 nm laser excitation than that excited by 808 nm laser.      In this work, we tested the stability of i-DNA in FBS by agarose gel analysis to mimic the in vivo environment ( Figure  R10). In addition, the i-motif DNA we used have cytosine-richened sequences (TTAACAAATTATATTATTCCCCTTTTCCCC, the C/G ratio is 30%), high content of guanine/cytosine bonds is considered to have a good stability.      presented as means ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by one-way ANOVA with Tukey's post hoc test were considered. Figure S21. Viability of the U87MG cells treated with different concentrations of ETPM and ECPM.